Instituto de Bioquímica Vegetal y Fotosíntesis IBVF – CIC Cartuja

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Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp. strain PCC 7120.
(American Society for Microbiology, 1994) Herrero Moreno, Antonia; Flores García, Enrique; Floriano Pardal, María Belen; Ministerio de Educación y Ciencia (MEC). España
A cloned DNA fragment from Anabaena sp. strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD. The expression of Anabaena sp. strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level. Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine. Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E. coli RNA polymerase sigma 70 consensus promoter. A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures
Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism
(American Society for Microbiology, 1995) Cruz Díaz, Jesús de la; Pintor Toro, José Antonio; Benítez Fernández, Concepción Tahía; Llobell González, Antonio; Comisión Interministerial de Ciencia y Tecnología (CICYT). España; European Commission (EC)
he enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes
A New Type of Asymmetrically Acting β-Carotene Ketolase Is Required for the Synthesis of Echinenone in the Cyanobacterium Synechocystis sp. PCC 6803
(Elsevier, 1997) Fernández González, Blanca María; Sandmann, Gerhard; Vioque Peña, Agustín; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
We have isolated, based on the knowledge of the complete genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803, an open reading frame (slr0088) similar to known bacterial carotene desaturases and have analyzed the function of the encoded protein. Surprisingly, this protein has no detectable desaturase activity with phytoene, hydroxyneurosporene, or ζ-carotene as substrates, but is rather a β-carotene ketolase that acts asymmetrically introducing a keto group on only one of the two β-ionone rings of β-carotene to generate echinenone. This is in contrast to the so far characterized β-carotene ketolases that act symmetrically, producing the di-keto carotenoid canthaxanthin from β-carotene without significant accumulation of echinenone. We have designated this new gene crtO The function of the crtO gene product has been demonstrated by 1) the biosynthesis of echinenone when the crtO gene is expressed in an Escherichia coli strain able to accumulate β-carotene, 2) the in vitro biosynthesis of echinenone from β-carotene with cell free extracts from E. coli cells that express the crtO gene, and 3) the absence of echinenone in a Synechocystis strain in which the crtO gene has been insertionally inactivated. The primary structure of the Synechocystis asymmetric ketolase bears no similarity with the known β-carotene ketolases. crtO is not required for normal growth under standard or high light conditions, neither is the photosynthetic activity of the crtO-deficient strain affected.
Site-directed Mutagenesis of Cytochromec 6 from Anabaena Species PCC 7119
(Elsevier, 1999) Molina Heredia, Fernando Publio; Díaz Quintana, Antonio Jesús; Hervás Morón, Manuel; Navarro Carruesco, José Antonio; Rosa Acosta, Miguel Ángel de la; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
A number of surface residues of cytochromec 6 from the cyanobacterium Anabaenasp. PCC 7119 have been modified by site-directed mutagenesis. Changes were made in six amino acids, two near the heme group (Val-25 and Lys-29) and four in the positively charged patch (Lys-62, Arg-64, Lys-66, and Asp-72). The reactivity of mutants toward the membrane-anchored complex photosystem I was analyzed by laser flash absorption spectroscopy. The experimental results indicate that cytochrome c 6 possesses two areas involved in the redox interaction with photosystem I: 1) a positively charged patch that may drive its electrostatic attractive movement toward photosystem I to form a transient complex and 2) a hydrophobic region at the edge of the heme pocket that may provide the contact surface for the transfer of electrons to P700. The isofunctionality of these two areas with those found in plastocyanin (which acts as an alternative electron carrier playing the same role as cytochromec 6) are evident.
Oxidizing Side of the Cyanobacterial Photosystem I
(Elsevier, 1999) Sun, J.; Xu, W.; Hervás Morón, Manuel; Navarro Carruesco, José Antonio; Rosa Acosta, Miguel Ángel de la; Chitnis, Parag R.; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
Photosystem I (PSI) interacts with plastocyanin or cytochrome c 6 on the luminal side. To identify sites of interaction between plastocyanin/cytochromec 6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein fromSynechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c 6 or an artificial electron donor was used. In contrast, cytochromec 6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c 6. Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wild-type PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins.
Site-directed Mutagenesis of Cytochromec 6 from Synechocystissp. PCC 6803
(Elsevier, 1999) Cerda Haynes, Berta de la; Díaz Quintana, Antonio Jesús; Navarro Carruesco, José Antonio; Hervás Morón, Manuel; Rosa Acosta, Miguel Ángel de la; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Dirección General de Investigación Científica y Técnica (DGICYT). España; European Union (UE); Junta de Andalucía
This paper reports the first site-directed mutagenesis analysis of any cytochrome c 6, a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochromec 6 from the cyanobacteriumSynechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c 6 are located in an acidic patch that may be isofunctional with the well known “south-east” patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochromec 6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c 6 acidic area, seems to be crucial for the interaction with the reaction center.
Conditional Expression of RNase P in the CyanobacteriumSynechocystis sp. PCC6803 Allows Detection of Precursor RNAs
(Elsevier, 2001) Tous Rivera, Cristina; Vega Palas, Miguel Ángel; Vioque Peña, Agustín; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
We have constructed a strain (CT1) that expresses RNase P conditionally with the aim to analyze the in vivotRNA processing pathway and the biological role that RNase P plays inSynechocystis 6803. In this strain, the rnpBgene, coding for the RNA subunit of RNase P, has been placed under the control of the petJ gene promoter (PpetJ), which is repressed by copper, cell growth, and accumulation of RNase P RNA is inhibited in CT1 after the addition of copper, indicating that the regulation by copper is maintained in the chimerical PpetJ -rnpB gene and that RNase P is essential for growth in Synechocystis. We have analyzed several RNAs by Northern blot and primer extension in CT1. Upon addition of copper to the culture medium, precursors of the mature tRNAs are detected. Furthermore, our results indicate that there is a preferred order in the action of RNase P when it processes a dimeric tRNA precursor. The precursors detected are 3′-processed, indicating that 3′ processing can occur before 5′ processing by RNase P. The size of the precursors suggests that the terminal CCA sequence is already present before RNase P processing. We have also analyzed other potential RNase P substrates, such as the precursors of tmRNA and 4.5 S RNA. In both cases, accumulation of larger than mature size RNAs is observed after transferring the cells to a copper-containing medium.
Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA
(Cold Spring Harbor Laboratory Press, 2001) Cruz Díaz, Jesús de la; Vioque Peña, Agustín; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Human Frontier Science Organization; Junta de Andalucía
tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3′ end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (ΔssrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of ΔssrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the ΔssrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics.
A Single Arginyl Residue in Plastocyanin and in Cytochrome c6 from the Cyanobacterium Anabaenasp. PCC 7119 Is Required for Efficient Reduction of Photosystem I
(Elsevier, 2001) Molina Heredia, Fernando Publio; Hervás Morón, Manuel; Navarro Carruesco, José Antonio; Rosa Acosta, Miguel Ángel de la; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c6 from Anabaena(Molina-Heredia, F. P., Dı́az-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999)J. Biol. Chem. 274, 33565–33570) and Synechocystis (De la Cerda, B., Dı́az-Quintana, A., Navarro, J. A., Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292–13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.
Light-induced absorption spectra of the D1–D2–cytochrome b 559 complex of Photosystem II: Effect of methyl viologen concentration
(Springer Verlag, 2001) Yruela Guerrero, Inmaculada; Torrado, Elena; Roncel Gil, Mercedes; Picorel Castaño, Rafael; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
The light-induced difference absorption spectra associated to the photo-accumulation of reduced pheophytin a were studied in the isolated D1–D2–Cyt b559 complex in the presence of variable methyl viologen concentrations and different illumination conditions under anaerobiosis. Depending on the methyl viologen/reaction centre ratio, the relative intensities of the spectral bands at 681.5±0.5, 667.0±0.5 and 542.5±0.5 nm were modified. The reduced pheophytin a located at the D1-branch of the complex absorbs at 681.7±0.5 nm, and at least two additional pigment species contribute to the Qy band of the difference absorption spectra with maxima at 667.0±0.5 and 680.5±0.5 nm. We propose the additional species correspond to a peripheral chlorophyll a and the pheophytin a located at the D2-branch of the complex, respectively. The blue absorbing chlorophyll at 667 nm is susceptible to chemical redox changes with a midpoint reduction potential of +470 mV. The Qx absorption bands of both pheophytins localised at the D2- and D1-branch of the D1–D2–Cyt b559 complex were at 540.7±0.5 and 542.9±0.5, respectively. The results indicated that the two pheophytin molecules can be photoreduced in the D1–D2–Cyt b559 complex in certain experimental conditions.
An evolutionary analysis of the reaction mechanisms of photosystem I reduction by cytochrome c6 and plastocyanin
(Elsevier, 2002) Rosa Acosta, Miguel Ángel de la; Navarro Carruesco, José Antonio; Díaz Quintana, Antonio Jesús; Cerda Haynes, Berta de la; Molina Heredia, Fernando Publio; Balme, Alexis; Murdoch, Piedad del Socorro; Díaz Moreno, Irene; Durán Díaz, Raúl V.; Hervás Morón, Manuel; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
Photosystem I reduction by the soluble metalloproteins cytochrome c6 and plastocyanin, which are alternatively synthesized by some photosynthetic organisms depending on the relative availability of copper and iron, has been investigated in cyanobacteria, green algae and plants. The reaction mechanism is classified in three different types on the basis of the affinity of the membrane complex towards its electron donor protein. The role of electrostatic interactions in forming an intermediate transient complex, as well as the structural and functional similarities of cytochrome c6 and plastocyanin are analysed from an evolutionary point of view. The proposal made is that the heme protein was first “discovered” by nature, when iron was much more abundant on the Earth's surface, and replaced by plastocyanin when copper became available because of the oxidizing conditions of the new atmosphere.
Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development
(Wiley, 2002) Muro Pastor, Alicia María; Valladares Ruiz, Ana; Flores García, Enrique; Herrero Moreno, Antonia; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Dirección General de Enseñanza Superior e Investigación Científica (DGESIC). España; Ministerio de Ciencia y Tecnología (MCYT). España
Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was, however, not dependent on HetR.
RNase P: Variations and Uses
(Elsevier, 2002) Gopalan, Venkat; Vioque Peña, Agustín; Altman, Sidney; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death
(The American Society for Biochemistry and Molecular Biology, 2003) Domínguez del Toro, Fernando; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier; Universidad de Sevilla. Departamento de Biología Celular; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis revealed PCD of aleurone cells in maize grains, although the process was delayed as compared with wheat. Aleurone cells undergoing PCD showed a rapid vacuolation with high lytic activity in the cytoplasm, whereas the nucleus, which adopted an irregular shape, appeared essentially intact and showed symptoms of degradation at the end of the process. A nuclease activity was identified localized in the nucleus of aleurone cells undergoing PCD, just prior to the appearance of DNA laddering. This nuclease was induced by gibberellic acid treatment and was not detected when gibberellin synthesis was inhibited or in gibberellic acid-insensitive mutants. This nuclease was activated by Ca2 and Mg2 , strongly inhibited by Zn2 , and showed optimum activity at neutral pH, resembling nucleases involved in apoptosis of animal cells.
The Different Large Subunit Isoforms of Arabidopsis thaliana ADP-glucose Pyrophosphorylase Confer Distinct Kinetic and Regulatory Properties to the Heterotetrameric Enzyme
(Elsevier, 2003) Crevillén Lomas, Pedro; Ballicora, Miguel A.; Mérida Berlanga, Ángel; Preiss, Jack; Romero Rodríguez, José María; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia y Tecnología (MCYT). España; Junta de Andalucía; Department of Energy. United States; Department of Agriculture. United States
ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according to the necessities for starch synthesis in a given tissue.
Detergent effect on cytochrome b559 electron paramagnetic resonance signals in the photosystem II reaction centre
(Wiley, 2003) Yruela Guerrero, Inmaculada; García Rubio, Inés; Roncel Gil, Mercedes; Martínez, Jesús I.; Ramiro Pascual, María Victoria; Ortega Rodríguez, José María; Alonso, Pablo J.; Picorel Castaño, Rafael; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
Detergent effect on Cytochrome b559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1-D2-Cyt b559 complex preparations. Various n-dodecyl-β-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1-D2-Cyt b559 complexes. Low spin heme EPR spectra were obtained but gz feature positions changed depending on detergent conditions. Redox potentiometric titrations showed a unique redox potential cytochrome b559 form (E ‘m = +123-150 mV) in all the D1-D2-Cyt b559complex preparations indicating that detergent does not affect this property of the protein in those conditions. Similar effect on Cytochrome b559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.
The Efficient Functioning of Photosynthesis and Respiration in Synechocystis sp. PCC 6803 Strictly Requires the Presence of either Cytochrome c6 or Plastocyanin
(Elsevier, 2004) Durán Díaz, Raúl V.; Hervás Morón, Manuel; Rosa Acosta, Miguel Ángel de la; Navarro Carruesco, José Antonio; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; European Union (UE); Ministerio de Ciencia y Tecnología (MCYT). España; Junta de Andalucía
In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the petE or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wild-type cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin.
Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions
(Wiley, 2004) Bernal Ibáñez, María; Roncel Gil, Mercedes; Ortega Rodríguez, José María; Picorel Castaño, Rafael; Yruela Guerrero, Inmaculada; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
Toxic Cu (II) effect on cytochrome b559 under aerobic photoinhibitory conditions was examined in two different photosystem II (PSII) membrane preparations active in oxygen evolution. The preparations differ in the content of cytochrome b559 redox potential forms. Difference absorption spectra showed that the presence of Cu (II) induced the oxidation of the high‐potential form of cytochrome b559 in the dark. Addition of hydroquinone reduced the total oxidized high‐potential form of cytochrome b559 present in Cu (II)‐treated PSII membranes indicating that no conversion to the low‐potential form took place. Spectroscopic determinations of cytochrome b559 during photoinhibitory treatment showed slower kinetics of Cu (II) effect on cytochrome b559 in comparison with the rapid loss of oxygen evolution activity in the same conditions. This result indicates that cytochrome b559 is affected after PSII centres are photoinhibited. The high‐potential form was more sensitive to toxic Cu (II) action than the low‐potential form under illumination at pH 6.0. The content of the high‐potential form of cytochrome b559 was completely lost; however, the low‐potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. The results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high‐potential and low‐potential forms of cytochrome b559, respectively.
Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium synechocystis sp. PCC 6803
(Elsevier, 2005) Ceballos Chávez, María; Vioque Peña, Agustín; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
Biosynthesis of transfer RNA requires processing from longer precursors at the 5′- and 3′-ends. In eukaryotes, in archaea, and in those bacteria where the 3′-terminal CCA sequence is not encoded, 3′ processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3′-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3′-side, respectively. The presence of the complete 3′-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.
NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c6
(Elsevier, 2005) Díaz Moreno, Irene; Díaz Quintana, Antonio Jesús; Molina Heredia, Fernando Publio; Nieto Mesa, Pedro Manuel; Hansson, Örjan; Rosa Acosta, Miguel Ángel de la; Karlsson, B. Göran; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
A structural analysis of the surface areas of cytochrome c6, responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c6 residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c6 were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins.

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